Preservation of food products



Patented Apr. 24, 1 951 2,550,255 PRESERVATION or FOOD PRODUCTS Lloyd B. Jensen, Chicago, Ill., assignor to Swift & Company, Chicago, 111., a corporation of Illinois No Drawing.

Original application April 5, 1946,

Serial No. 659,881. Divided and this application November 18, 1950, Serial No; 196,519

' The present invention relates to the preparation of a potent antibiotic substance, and it has to do more particularly with the concentration of an antimicrobial substance of plant origin.

For some time it has been known that some pathogenic bacteria are destroyed when soil is inoculated therewith. This destruction of bacteria is due to certain bacteria or antibacterial substances contained in the soil. It is also well known that the putrefying bacteria responsible for food spoilage do not ordinarily develop in living tissues of plants and animals. The presence of antibodies in live animal tissues and of antibiotic substances in plants are thought to play an important part in establishing immunity in living organisms against microbes harmful to the organism. From time immemorial it was thought that certain medicines of herbal origin were of some therapeutic value. Pursuant to this knowledge, microbiologists have been seeking to isolate useful microorganisms and prepare antibacterial substances from natural sources which antibacterial materials, although disassociated from their environment, might be capable of combatting microorganisms harmful to man.- Although living matter is known to contain substance of an antibacterial nature, it is impossible to predict whether or not portions of a living organism, for example a plant, will yield an effective antibacterial substance when subjected to known methods of separation, concentration, extraction, or purification; or if once isolated whether or not the antibacterial substance will be beneficial. As a result of this search for antibacterial agents, naturally occurring antibiotic substances have been prepared from plants, animal tissues, molds, antinomyces, yeast and bacteria, for example, penicillin from the mold Penicillium notatum, and gramicidin. from certain soil bacteria. It is known that some of these substances are in themselves toxic, are for the most part heat labile, and while they might be lethal in small concentrations to certain types of bacteria, they are, on the other hand, ineffective against other types or strains of bacteria.

An object of the present invention is to provide a potent antibiotic substance of plant origin.

A further object of the invention is to provide an antibiotic substance which is not toxic.

A still further object of the invention .is to provide an antibiotic substance which is heat stable.

Yet another object of the invention is to provide an antibiotic substance which may be employed as an effective preservative for food products. I

6 Claims. (01. 99-150) Still another object of the invention is to provide an antibiotic material which is capable of inhibiting mold growth.

Other objects and advantages of theinvention will be apparent from a description of the invention given below.

The present invention contemplates broadly the preparation of an antibiotic substance found to be naturally associated with tissues of the tree Persea gratissima, or Pers'ea americana, commonly known as the avocado tree, including the fruit thereof. I have discovered that especially the pit or pith of the avocado fruit or pear yields an antibiotic substance of great potency, to be hereinafter more fully described. I have also found that the antibiotic substance produced from the avocado pit is lethal to those microorganisms largely responsible for clostridial and bacillary food spoilage, and staphylococcus food poisoning and therefore may be used advantageously in the preservation of food products. My antibiotic substance has been found to destroy gas-forming bacilli of the genus Bacillus, such as Bacillus subtilis (var. m'ger) molds of the genera Aspergillus andPenicillium, such as Aspergillus glaucus, and Penicillium notatum; the

bacilli of the genus Clostridium, such as C'ZOStTidium sporogenes, the staphylococci, such as Staph-' ylococcus aureus. I have also found my antibiotic substance to be lethal against the bacterial species Achromobacter perolens, the organism responsible'for the production of a musty odor in eggsknown as hay eggs .While the. antibacterial substance produced from the avocado-pit is extremely effectiveagainst theaforesaid organisms, I have found that other'organisms, for example, Escherichz'ajcolz', .Peniczllium camembertz', the Pseudomonas fluorescens and Pseudomonas syncyanea are more resistant thereto. It would be an extremely arduous, if not animpossible, task to ascertain the response of all the existing species and varieties of microorganisms to the herein described antibiotic material but the effects of this material against the microorganisms hereinabove mentioned illustrate the commercial and medical values thereof.

The invention contemplates treating a mixture of ground avocado piths or pits with a suitable solvent to recover the antibiotic substance from the avocado material. A colored powder remains after evaporating off the solvent and may'advantageously be refined by treating again with a suitable solvent, such as ethanol.

The following examples illustrate preferred embodiments of the invention, and the tests falling thereunder illustrate the potency of the material 3 obtained in accordance with the processes of the examples.

Example I.250 g. of avocado piths or pits (brand Calavo) were peeled and ground finely in a small hand meat grinder. The pits were cream colored with a slight yellowish tinge when whole, but upon grin'ding'the piths became redorange in color and emitted a faint pleasant odor somewhat like a mixture of cinnamon and pine needles. The ground piths were covered with 500 cc. of acetone and held in a flask for 24 hours, the flask being shaken at intervals. The acetone solution was removed from the, pith by filtration and the acetone was distilled off under a 29 inch vacuum at 45 C. The yellowish brown residue was dissolved in 50 cc. of 95 per cent 'ethanol, so as to remove the possibility of solvent toxicity and to prevent contamination. The alcohol alsoprovides means for accurately measuring the en th o he a t b ot c ub ta c thu th use of 50 cc. of alcohol makes possible the establishment of an arbitrary standard unit of antibiotic substance, that is, 1 ml. of alcoholic extract to represent g. of original plant material Two tests were then performed to evaluate the efiectiveness of the product obtained in accordance with the process described in Example I.

Test 1 .One hundred milliliters of nutrient agar (obtained from Difco Laboratories, Detroit, Michigan) were inoculated with 0.2 ml. of a 20-hour broth culture of a food poisoning strain of Staphylococcus aureus, which had been tested on Ma cacus rhesus monkeys and found to produce enterotoxin. Plates were poured with this agar. A hole was bored in the center of, the hardened agar so as to form a cup in the agar plates. The cups. were then filled with aqueous dilutions of the antibiotic product (1 ml. of the product representing 5 g. of pith) and were incubated at 37 C. for 4 days. Although the inoculum was rather'heavy so as to prevent good diffusion thereof through the agar, the areas of inhibition indicated in the following table were obtained.

Table I at 1:150 dilutions, 5 mm. away; 1:100 dilution,

4 mm. away, etc. Presumably, the clear zone of inhibition characterized by the absence of bacterial growth represented the extent to which the antibiotic material had'difiused through the agar.

The agar cup plate method is open to objec-= tions in evaluating the potency of antibiotic or other antibacterial substance because the method depends in part upon rate of difiusion of the substance through the agar. Serial dilution methods innutrient broth tubes, as described below, were then employed to demonstrate antibiotic action on bacteria.

' Test 2.In this test the potency of the antibiotic substance obtained from Example I was determined by serial dilutions of the extract in cc. broth tubes and then inoculated with bacteria. Dilutions of the extract were made in broth tubes from lzlo to 1230000 and inoculated with large loops of 24-hour broth cultures of a food poisoning strain of Staphylococcus du're'us (50,000 cells). The test organism was inhibited at a dilution of 1:5000 after 7 days incubation at 37 C. After 7 days all of the tubes showing no growth were used as inocula in nutrient agar plates, 0.1 ccvof the'material in the broth tubes being transferred to each agar plate. No growth resulted in 3 days from these transfers, which shows that the antibiotic substance had killed the test bacteria in a 1:5000

dilution.

Example II 340 g. of avocado piths (Rancho- La Habra brand) were ground under acetone andcovered with acetone for 24 hours. The solvent was evaporated off under 27- inches of vacuum (water pump) at 40 C. and the residue taken up in 68 ml. of pure ethyl alcohol, so that each ml. of alcoholic solution represented 5 g. of piths.

The effectiveness of the bactericidal properties of the product obtained by means of the process described in Example II, was then tested according to the method of Test 2 of Example I; that is, serial dilutions of 10 cc.-broth tubes were made, each tube inoculated with a large loopful of a broth culture of Staphylococcus laureus incubated for 24 hours at 37 C. The inoculated tubes were then allowed to incubate at37 C. and were observed for growth at the end of 24, 48, and 120'hours. The following table gives the dilutions and results as to bacterial growth corresponding therewith after 24, 48, and 120 hours, the minus sign indicating no growth, the positive sign the presence of growing staphylococci.

Table II Dilutions in Broth 24 hours. 48 hours. hours.

' cating that the antibiotic substance'was an eftested for potency against other species of bacteria indicating no growth, the positive sign indicating and also molds. It was found that Clostridium sporogenes (National Canners Association strain #3679) was killed in a 1: 10,000 dilution in Difco peptone colloid tubes; species of genus Bacillus in 1:500 dilution in Difco nutrient broth; Achromobacter perolens, Aspergillus glaucus and Penicillium notatum also in 1:500 dilution, and another strain of Staphylococcus aureus 1:10,000 dilution.

Although I have found that the pits of the fruit of. the avocado tree are especially rich in a powerful antibiotic substance, other parts of the tree, such as the leaves, stalk, roots and meat of the fruit contain the antibacterial substance to a leser extent, but sufliciently to show bacteriostatic, or antiseptic, properties.

The antibiotic substance contained in Persea gratissimd is soluble in a number of solvents.

Suitable solvent media are the polar solvents, a

such as acetone, methyl ethyl ketone, ethyl, propyl and butyl alcohols, dioxane, ethyl acetate; chlorinated solvents, such as carbon tetra-chloride, trichloroethylene; hydrocarbons, such as pentane, hexane, etc. Since the antibiotic material isolated from the avocado plant is heat stable, solvent treating temperatures may range from room temperatures to boiling temperature of the solvent. The type of solvent chosen aifects the color of the dried product. For example, acetone gives a brownish red product; hexane and trichloroethylene, a yellow material; ethyl alcohol, methyl ethyl ketone, ethyl acetate, a red substance, etc.

It is obvious that many variations in the solvent treating process may be advantageously employed. To illustrate, the treatment may be carried out in' one step, for example, with ethanol;

mixtures of solvents and different temperatures may also be advantageously employed to increase efficiency. I have found it preferable to process the antibiotic substance contained in the avocado pit by a two-stage solvent process, involving, first, treatin the substance by means of a primary solvent which is subsequently removed, and then, second, taking up the resulting residue with another solvent, so .as to form a solution, which is a convenient form of using the product. I have found that acetone is a very effective primary solvent in producing the antibiotic substance from the plant tissue. After evaporating off the acetone, the preferred solution is made with ethanol. By using alcohol I may utilize its well-known properties, such as nontoxicity and ability to prevent contamination.

I have found the antibiotic substance to be slowly soluble in distilled water. When the alcoholic extract of pith is added to water or broth,

a heavy brownish-yellow precipitate is formed immediately. If the alcoholic solution in broth or water is centrifugalized and dilutions made in broth tubes of both supernatant liquid and sediment which are then inoculated with bacteria, both supernatant liquid and sediment inhibit bacterial growth. For example, a 1:10 solution of the alcoholic solution in broth was centrifugalized 30 minutes at high speed and dilutions made in broth tubes of both supernatant liquid and sediment. The tubes (10 ml.) were each inoculated with a loopful of a -hour broth culture of food poisoning Staphylococcus aureus and incubated at 37 C. Bacterial growth at 1:100, 1:500, 1:1-000, 1:2000, 1:5000, and 127500 dilutions of the extract were observed after 24 and 48 hours. The following table lists the results, the minus sign bacterial growth.

The data tabulated above show that the precipitate is slowly soluble in water and is a powerful antibiotic substance.

'In the processing of food products, a serious problem is that of food preservation, and although great strides have been made in improving the quality and keeping time of various food products by refrigeration, canning, curing, plant sanitation, etc, the problem of the preservation of food requires the constant attention of the food processor to improve long-established methods of treating food and to devise new means of food control.

Food spoilage is due principally to microbial action, and the microorganisms responsible for food deterioration are numerous. The sporing genera, Clostridium and Bacillus, for example, when contaminating food, cause food spoilage. Molds of the genera Penicillium and Aspergillus are also responsible for much food spoilage. In addition to food spoilage, a serious concern of the food handler is food poisoning, which is often of an insidious nature because food products contaminated with harmful bacteria often appear organoleptically sound and wholesome. Among the food poisoning bacteria, the most common food poisoner is the'staphylococcus. This organism is widespread in nature, and is believed to be responsible for over 90 per cent of food poisoning cases in the United States, in addition to its being responsible for many infections in man and animals.

The antibacterial substance of the present invention may be used to retard or prevent microbial growth in various food products. This material may advantageously be employed in treating meat products,,such as hams, sausages, and canned meats; filled pastries, such as cream puffs, chocolate eclairs, custards, etc. canned vegetables to prevent swellings due, for example, to organisms of the genus Bacillus.

The invention will be more fully understood from the following description of a method of using my antibiotic substance, in connection with the curing of hams.

Within recent years there has been developed a curing process for meats and particularly hams known as a quick-cure which requires only a fraction of the time which was normally required by the old processes. Essentially the quick-cure process consists in pumping the pickling solution under pressure into the vascular system of the meat. By this means a ham can be cured in a period of time ranging from fifteen to eighteen days, rather than the sixty to ninety days re- 7 tens'ive c'omine'rcia'luse -of-the quick-curing method an attendant serious healthproblem. The hams and other meatscuredby the older immersion processes were extremely resistant to the development of pathogenic bacteria, of which various representatives of the Staphylococci may be mentioned as typical, and the general public has come to reply on this property in its eating and storage habits.- Hams processed by quickcuring, on the other hand, have resulted in a-..

meat product which, though-highly satisfactory when fresh, is not nearly so resistant to. these organisms, and numerous cases-of food poisoning have resulted from the general publics unfami liarity with this fact.

As hereinbefore mentioned, we have found that the antibiotic reagents herein described islethal to a number 'of microbes in dilution of 1:5000, based on the arbitrary unit that 1 ml. of solu tion is equivalent to 5 g. of the "source material, or

original pith. Therefore, the addition of one part of the solution to 5000 partsof the conventional pickling solution will result in a meat product cured therewith which has a very marked increase in resistance to the development of pathogenic organisms.

The following is an example of a curing pickle :stock, which may be usedin a process for quickcuring ham when provided with antibiotic sub- .stance in the proportions outlined above; that is, the antibiotic substance is added in an amount .such as to effect a 1:5000 :dilution of the antibiotic material,.based onlthe arbitrary standard herein described (1 .m1.=5 g. of pith).

To 100 gallons of a.90"..Salometer solution there .are added:

"7 lbs. sodium nitrate '10 '02. sodium nitrite 120 lbs; sugar 75.7 cc. antibiotic solution The following experiments will further illustrate the practical application of my invention. An amount of pickle was inoculated with a large number of a food poisoning type of Staphylococcus am'eus organisms which would represent the most unfavorable conditions which could possibly be encountered. The pickle was then separated into two equal portions, and to one portion sufficient amount of avocado product was added to form a 1:5000 dilution of the product. To the other portion of pickle no antibiotic substance was added. Twelve hams were then cured 'by the quick-curing method-six of'them with pickle containing the antibiotic substance and six with pickle without the substance. then smoke according to ordinary processes. The hams were incubated at 99 F. and at the end of two weeks the six hams containing the antibiotic treated hams did not harbor viable Staphylococci, while the other hams showed the presence of viable Staphylococci.

Luncheon meat was cured according to conventional methods in which the antibiotic substance was present in a 1:5000 dilution. The

meat product was placed in 6 sixpou'nd cans and A retorted to an inside temperature of 155 F. and also incubated at 99 F. After 30 days of incubation, no swelling of the cans could be observed. The control cans swelled in one week.

A dry acetone solution of product from 60 g.

The hams were of avocado pith was 'fed to --four white rats -for six days. "The animalsconsumed 600 'g.-'of ground meat to'which wasadded 1per cent of the'product. The a'nimals showed'no symptoms of'toxicity and there were-no losses in weight.

'This application is 'a divisional application of U. S. Patentapplicatio'n Seria1No.'659,881,filed April 5,1946.

Obviously, many modifications and variations of the invention, as hereinbefore set forth; maybe made without departing from the spirit and scope thereof, and therefore only such limitations shouldbe imposed'as arev indicatedin'the appended claims.

I Iclaim:

1. In the process of treating food products, the step of adding to a food product a small'amount of an antibiotic substance derived from the. tree Persea gratissima. by treatment with a nonaqueous organic solvent torpreserve said food product against pathogenic bacteriaresponsible for food poisoning and spoilage.

2. In the process of treating food products, the step of adding to a food product a small amount of an alcoholicsolution of antibiotic substance derived from the tree Perseagratz'ssima to preserve said food product against pathogenicbacteria responsiblefor food poisoningand spoilage.

3'; In the processor quick-curing meats, wherein a pickling solution is injected. into said meat, the step of adding to saidpickling solution a small amount of the antibiotic substancederived from the root of the tree Persea gratz'ssima by treatment with a non-aqueous organic solvent whereby the growth; of pathogenicrbacteria responsible for food spoilage and poisoning inthe meat is substantially retarded.

4. In the process 'ofpreparing pastries containing fillings subject to; spoilage by pathogenic bacteria, the step. of adding to the said fillings a small amount of the antibiotic substance derived from the root of the tree Persea. gmtissima by treatment with a non-aqueous organic solvent whereby the-growthofpathogenic bacteria in the said fillings is substantially retarded.

.5. A meat pickle including a small amount of an antibiotic substance derived from .the root of the tree Persea g'ratz'ssz'maby treatment with a non-aqueous organic solvent.

6. A cream filling forz-pastries including a small amount of an antibiotic substance of the root derived from the tree Persea-gratissima'by treatment with a non-aqueous organic solvent.

LLOYD B. JENSEN.

REFERENCES CITED The following references .are of recordinthe file of thispatent:

UNITED STATES PATENTS lName Date Hall May 17, 1938 OTHER. REFERENCES Number 

1. IN THE PROCESS OF TREATING FOOD PRODUCTS, THE STEP OF ADDING TO A FOOD PRODUCT A SMALL AMOUNT OF AN ANTIBIOTIC SUBSTANCE DERIVED FROM THE TREE PERSEA GRATISSIMA BY TREATMENT WITH A NONAQUEOUS ORGANIC SOLVENT TO PRESERVE SAID FOOD PRODUCT AGAINST PATHOGENIC BACTERIA RESPONSIBLE FOR FOOD POISONING AND SPOILAGE. 